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From the Departments of Ophthalmology,*
Neuroscience,
and Molecular Biology and
Genetics,
The Johns Hopkins University School
of Medicine, Baltimore; and the National Cancer
Institute,§
National Institutes of Health,
Bethesda, Maryland
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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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In the eye, there is low level constitutive expression of PDGFs in perivascular cells, ganglion cells, and in the retinal pigmented epithelium (RPE).4,5 It appears to play a role in recruitment of pericytes and astrocytes into the retina, and may support the survival of these cells in adult retinas.6,7 After retinal detachment there is marked up-regulation of PDGF expression in the RPE.5 The retina can be reattached by surgery, but in roughly 10% of cases, scar tissue made up predominantly of RPE and glial cells forms in the vitreous and on the surfaces of the retina and results in traction that redetaches the retina.8 There is prominent expression of PDGFs in the cells that participate in the scarring. This disease process is called proliferative vitreoretinopathy, the most common cause of failure of retinal reattachment surgery, and PDGFs have been implicated in its pathogenesis.9,10 But PDGF also contributes to regulation of development11-14 and expression of PDGF in neurons suggests possible neurotrophic and/or gliotrophic effects.4,15
PDGF is a dimer made up of the products of the PDGF-A and
PDGF-B genes, resulting in three isoforms, PDGF-AA,
-AB, and -BB. Similarly, two gene products result in three types of
dimeric receptors, PDGF receptor (PDGFR)-
, -ß, and
-ßß.16
PDGFR- binds both PDGF-A and -B, while
PDGFR-ß binds only PDGF-B. Therefore, PDGF-A is more selective than
PDGF-B and, theoretically, it might affect fewer cell types in a given
tissue than PDGF-B depending on the expression of and ß receptors
on various cell types.
At least two cell types appear to have very selective expression of
PDGFR isoforms. Glial precursor O2-A progenitor cells from the optic
nerve express PDGFR-, but not PDGFR-ß,17
while
pericytes express PDGFR-ß, but not PDGFR-.6
Also, it
has been suggested, based on in vitro data, that
endothelial cells participating in angiogenesis may express
PDGFR-ß.18
Because each of these cell types is located
in the retina, the retina provides a good model system to explore
similarities and differences in the activities of PDGF isoforms.
Recently, we coupled the cDNA of human PDGF-A to the bovine rhodopsin promoter and generated transgenic mice (rho/PDGF mice) with photoreceptor-specific expression of PDGF-A.19 These mice developed a fairly subtle phenotype due to glial infiltration of the inner retina. The ectopic glial cells conferred a striking resistance to oxygen-induced retinal vascular nonperfusion and prevented the development of neovascularization. In this study, we used gain-of-function transgenic mice that express PDGF-B in photoreceptors to compare the effects of PDGF-B in the retina to those of PDGF-A and to determine whether these mice would exhibit a phenotype like that seen in patients with proliferative retinopathies.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A 907-bp cDNA fragment of human PDGF-B, including 117
bp of 3' untranslated sequence, the entire coding region, and 68 bp of
untranslated 5' sequence, was cloned into a plasmid containing the 2.2
kb HindIII/NaeI fragment from the bovine rhodopsin
promoter.20
The plasmid also contained an intron and a
polyA addition site derived from the mouse protamine gene and a
eukaryotic consensus ribosomal binding site. After transformation, a
clone with correct orientation was selected. DNA was double CsCl
purified and cut with EcoRI to provide a 3579-bp fusion gene
(Figure 1)
. The fusion gene was purified
and transgenic mice were generated using established techniques as
previously described.20
The fusion gene was injected into
the pronuclei of B6AF1 (female) x C57BL/6J
(male). All offspring were backcrossed with C57BL/6J mice.
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. Retinal Reverse Transcriptase PCR
At appropriate time points, mice were sacrificed, eyes were
removed, and retinas were dissected. Retinal RNA was isolated using the
guanidine isothiocyanate method as described by Chomczynski and
Sacchi.21
Reverse transcription was carried out with
0.5 µg of total RNA, reverse transcriptase (SuperScript II, Life
Technologies, Gaithersburg, MD), and 5.0 µM oligo d(T) primer.
Aliquots of the cDNAs were used for PCR amplification with primers for
the hPDGFB/mP1 fusion gene that amplify across an
intron-exon border, P3 (5'-ATAGACCGCACCAACGCCAACTTC-3') and P4
(5'-TGTGGCGAGATGCTCTTGAAGTCTGGTA-3') (Figure 1)
. The expected PCR
products for the hPDGFB/mP1 fusion gene fragment from
genomic DNA and mRNA are 787 bp and 693 bp, respectively. Titrations
were performed to ensure that PCR reactions were carried out in the
linear range of amplification. Mouse S16 ribosomal protein primers
(5'-CACTGCAAACGGGGAAATGG-3' and 5'-TGAGATGGACTGTCGGATGG-3') were used
to provide an internal control for the amount of template in the PCR
reactions.
Northern Blot Analysis
RNA blot hybridization analysis was done as previously described5 using 10 µg of total retinal RNA. The 842 bp BamHI fragment of hPDGFB or the 598-bp BamHI fragment of hVEGF were labeled with 32P by hexanucleotide random priming and used as cDNA probes. Hybridization temperature was 65°C and the membrane was washed twice for 60 minutes at room temperature in 2x SSC, 0.1% SDS, followed by a 15 minute wash at 58°C in 1x SSC, 0.1% SDS, and a final 15-minute wash at 65°C in 0.5x SSC, 0.1% SDS.
Immunohistochemistry for PDGF-B
Transgene-positive and littermate control mice were sacrificed at various time points, and their eyes were removed and frozen in optimal cutting temperature medium (OCT, Miles Diagnostics, Elkhart, IN). Ten-micron sections were cut and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS). The sections were washed and immunohistochemical staining was performed as previously described5 with a 1:50 dilution of a monoclonal antibody that recognizes PDGF-B.22 Specificity of staining was assessed by substitution of nonimmune serum for primary antibody and by preabsorption of primary antibody with antigenic peptide as previously described.23
Evaluation of the Retinal Phenotype of Transgenic Mice
At various time points, mice were sacrificed and eyes were
snap-frozen in OCT or fixed in 10% buffered formalin and embedded in
paraffin. Frozen or paraffin sections were stained with hematoxylin and
eosin (H&E), histochemically stained with biotinylated Griffonia
simplicifolia isolectin-B4 (GSA; Vector Laboratories, Burlingame,
CA), or immunohistochemically stained with a 1:1000 dilution of a
rabbit polyclonal antibody to glial fibrillary acidic protein (GFAP;
Dako, Santa Barbara, CA), a 1:50 dilution of rabbit polyclonal
antibodies to -smooth muscle actin (Biogenix, San Ramon, CA), a 1:40
dilution of rabbit polyclonal antibody to vascular endothelial growth
factor (VEGF; Santa Cruz Biotechnology, Santa Cruz, CA), or a 1:200
dilution of a rabbit polyclonal antibody directed against
platelet-endothelial cell adhesion molecule (PECAM; Santa Cruz
Biotechnology). PDGF-B was visualized with HistoMark Red (Gaithersburg,
MD) according to the manufacturer’s instructions and other antigens
were visualized with diaminobenzidine (Research Genetics, Huntsville,
AL).
Staining with biotinylated GSA (Vector Laboratories), which selectively binds to vascular cells, was done as previously described.24 Slides were incubated in methanol/H2O2 for 30 minutes at 4°C, washed with 0.05 mol/L Tris-buffered saline (TBS), pH 7.4, and incubated for 30 minutes in 10% normal porcine serum. Slides were rinsed with 0.05 Mol/L TBS and incubated 1 hour at 37°C with biotinylated lectin. After being rinsed with 0.05 Mol/L TBS, slides were incubated with avidin coupled to peroxidase (Vector Laboratories) for 45 minutes at room temperature. After being washed for 10 minutes with 0.05 mol/L Tris buffer, pH 7.6, slides were incubated with diaminobenzidine (Research Genetics) to give a brown reaction product, and mounted with Cytoseal (Stephens Scientific, Riverdale, NJ).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six independent lines that incorporated the rhodopsin
promoter/PDGFB fusion gene (Figure 1)
were obtained (designated
rho/PDGFB1–6). The founders were backcrossed with C57BL/6J
mice to establish transgenic lines. Mice that were heterozygous at the
transgene locus were used in all analyses.
Expression of PDGF-B mRNA in the Retinas of Transgenic Mice
At postnatal day 14 (P14), RT-PCR, using total retinal RNA as
template and primers specific for transgene mRNA, showed good
expression in lines rho/PDGFB1–4, barely detectable
expression in line 5, and no detectable expression in line 6 (Figure 2A)
. A time course in mice from line 1
showed transgene mRNA was first detectable at P5 or P6, increased to a
steady state level by about P10, and was maintained through at least 3
months after birth, the longest time point examined (Figure 2B)
. Northern analysis also suggested that steady state levels of
mRNA were reached by day 10, and there appeared to be similar levels of
expression in lines 1, 2, and 3 (Figure 2C)
.
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Immunohistochemistry with an antibody that specifically recognizes
PDGF-B showed reaction product in photoreceptors, with an intense band
of staining in the region of photoreceptor terminals (Figure 3)
. Similar localization was seen for
PDGF-A in several lines of rho/PDGFA mice,19
suggesting the possibility of polarized secretion. Although
rho/PDGFB mice show the greatest staining for PDGF-B in
photoreceptors, there is also increased staining for PDGF-B in the
inner retina compared to wild-type mice, suggesting that PDGF-BB
diffuses through the retina and is accessible to cells in the inner
retina.
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For each of the 6 transgenic lines, histopathological evaluation
was done between 3 and 9 months of age on 8 to 10 mice selected from 3
generations. All mice from lines 1–4 showed traction detachments of
the retinas in both eyes, whereas mice from lines 5 and 6 had normal
appearing retinas with no detachments, presumably due to the low
expression levels of transgene-derived PDGF-B in the latter two lines.
In many instances, the retina became adherent to the back of the lens,
forming a retrolental mass. There was striking degeneration of the
detached retinas and prominent epiretinal and subretinal cellular
membranes (Figure 4)
. The traction
detachments in rho/PDGFB mice are similar to the traction retinal
detachments seen in humans with proliferative vitreoretinopathy, in
which there are tractional membranes made up primarily of retinal
pigmented epithelial (RPE) cells and retinal glial cells, or to the
traction retinal detachments seen in retinopathy of prematurity, in
which there is a prominent vasoproliferative component in addition to
proliferation of RPE and glia. To explore why overexpression of PDGF-B
in the retina leads to traction retinal detachment, a detailed
investigation of the cellular involvement and sequence of events was
performed primarily in lines 1 and 2.
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Astrocytes are small glial cells that in adult mouse retinas are
normally found in the nerve fiber layer and adjacent to retinal blood
vessels. Muller cells are large glial cells extending from the inner to
the outer surface of the retina. Developing astrocytes are known to
have PDGFR-s and are very responsive to PDGFs for at least 2 weeks
after birth.25
They migrate from the optic nerve into the
posterior portion of the inner retina and then spread anteriorly until
they populate the entire nerve fiber layer. They associate with blood
vessels in the nerve fiber layer and are visualized by
immunohistochemical staining for glial fibrillary acidic protein
(GFAP). At P6, which is soon after transgene expression begins,
rho/PDGFB1 or -2 and wild-type mice showed
identical patterns of sparse GFAP-positive astrocytes at the inner
surface of the retina (Figure 5
, P6+ and
P6-), but by P7, GFAP staining along the inner surface of the retina
was increased in transgenics compared to wild-type mice. The difference
was more pronounced at P8 when there was clearly a multilayered
collection of astrocytes in transgenics. At P9 and P10, in addition to
continued sparse staining at the retinal surface, wild-type mice showed
focal areas of GFAP staining in the inner nuclear layer, presumably due
to migration of astrocytes into the retina along with penetrating
retinal vessels. In contrast, rho/PDGFB1 and -2
mice showed a thick carpet of astrocytes on the surface of the retina
and cords of invading cells extending into the inner nuclear layer that
were quite prominent by P10. At P12 and beyond, there was often
evidence of retinal folding with focal areas of retinal detachment that
progressed to total retinal detachment at later time points.
Perturbations of the retina, including detachment, often result in
increased expression of GFAP in Muller cells; this is illustrated by
the radial streaks of staining seen at P14 and beyond in transgenics
(Figure 5
; P14, P21, and P28). The carpet of astrocytes along the
retinal surface became more compact and less prominent at later time
points.
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Pericytes have PDGFR-ßs,4,6 and PDGF-BB stimulates their proliferation.26 Also, there is some suggestion that endothelial cells may express PDGFR-ßs under certain conditions.18 Therefore, we sought to determine whether pericytes and endothelial cells participate in the proliferative response in rho/PDGFB mice. GSA lectin selectively binds vascular cells and has been suggested to be an endothelial cell-specific marker,27,28 but we have recently demonstrated that it binds to both endothelial cells and pericytes.29
There was identical staining for GSA on the surface of the retina in
wild-type and transgenic mice at P6, which is around the time of onset
of PDGF-B expression in transgenics (Figure 6)
. This represents the developing
retinal vessels on the surface of the retina. By P7 there was more GSA
staining along the inner surface of the retina in transgenics compared
to wild-type mice, and the difference was more pronounced at P8 when
there was clearly a multilayered collection of vascular cells in
transgenics. At P9, wild-type mice show penetrating retinal vessels
that form the intermediate and deep capillary beds that assume the
appearance seen in normal adults between P10 and P12. In transgenics,
the formation of the deep capillary beds was impaired. On P9, there
were many more vascular cells on the surface of the retina in
transgenic compared to wild-type mice, but fewer vascular cells
had migrated into the retina. At P10, there were thick, finger-like
projections extending into the inner retina from a multilayered carpet
of cells on the retinal surface. The invading cords of vascular cells
did not appear to be organized into vessels. At P12, much of the inner
retina was infiltrated and replaced by GSA-positive cells. At P14 and
later time points, there appeared to be some involution and contraction
of the vascular cells, resulting in focal areas of retinal detachment
that progressed and resulted in total retinal detachment.
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. Mice
from line 5, which had barely detectable transgene expression, showed
the mildest phenotype with some development of the deep capillary bed
and minimal increase in GSA-positive cells in the inner retina. A
deficiency of normally perfused retinal vessels was confirmed in whole
mounts of fluorescein-labeled dextran-perfused retinas from line 1
(Figure 8)
. The capillary network was
much less dense in transgenics compared to wild type mice, and
there were several moderate-sized areas of nonperfusion (Figure 8B
,
asterisks). As expected from the disorganized appearance of the
vascular cell proliferation seen in retinal sections, whole mounts of
perfused retinas failed to show definite neovascularization, but did
show occasional vascular malformations (Figure 8B
, arrows).
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. Staining for PECAM showed thin
tube-like structures within the thick, PECAM-negative cell mass on the
surface of the retina (Figure 9, A, D, and G)
, whereas retinas stained
for smooth muscle actin looked very much like GSA-stained retinas,
showing large groups of cells that were not organized into recognizable
structures. Collectively the immunohistochemical data suggest that the
proliferating ectopic cells in the retinas of rho/PDGFB mice
consist of large accumulations of pericytes and astrocytes surrounding
small tube-like structures made up of endothelial cells.
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The prominent participation of vascular cells in the proliferative
response of rho/PDGFB mice caused us to compare levels of
VEGF mRNA in transgenic and wild-type mice. VEGF was
increased in the retinas of transgenics compared to wild-type mice at
P21 (Figure 2C)
. This is likely to be due at least in part to poor
development of the intermediate and deep capillary beds resulting in
areas of nonperfused retina (Figure 8)
. Based on the results of
GSA-staining, it is likely that the onset of retinal ischemia occurred
around P10 (Figure 6H)
. Ectopic astrocytes and pericytes may have also
contributed to increased VEGF mRNA in transgenics, since
both of these cell types have been demonstrated to produce VEGF.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is intriguing that two members of the PDGF family with substantial homology have such markedly different effects in the retina. The basis for the difference appears to be the broader stimulatory effects of PDGF-B compared to PDGF-A. Rho/PDGFB mice show increased numbers of astrocytes, pericytes, and endothelial cells, whereas rho/PDGFA mice show only increased astrocytes. Other models, including mice with oxygen-induced ischemic retinopathy33 and rho/VEGF transgenic mice,34 show increased numbers of endothelial cells and pericytes in the retina, and they also have different phenotypes from that of rho/PDGFB mice. In the former, the endothelial cells and pericytes are increased in normal proportions and they are organized into blood vessels, whereas in rho/PDGFB mice, there are many more pericytes than endothelial cells, and they form disorganized sheets of cells. One possible interpretation is that in rho/VEGF mice and mice with oxygen-induced ischemic retinopathy, the primary stimulus, VEGF, causes migration and proliferation of endothelial cells, which in turn recruit pericytes, probably by production of PDGF-B. In rho/PDGFB mice, the primary stimulus, PDGF-B, causes excessive proliferation of pericytes and astrocytes, which in turn recruit endothelial cells, probably by the production of VEGF. The relative difference in amount of PDGF versus VEGF and/or the reverse order of their production cause major phenotypic differences resulting in organization of proliferating endothelial cells and pericytes into blood vessels in rho/VEGF mice and mice with oxygen-induced ischemic retinopathy, whereas in rho/PDGFB mice, the proliferating cells form sheets and cords with limited organizational structure. It is tempting to speculate that in rho/PDGFB mice, the massive proliferation of pericytes, which are cells with a highly developed contractile apparatus, and their failure to organize into vessels, which may serve to harness their contractile function, results in contractile sheets of cells that foreshorten the retina, initially causing folds in the outer retina and ultimately progressing to total retinal detachment.
Cultured vascular endothelial cells express PDGFR-ßs during tube formation,18 but in situ hybridization demonstrates PDGFR-ß mRNA in murine brain pericytes, but not endothelial cells, including those in new vessel sprouts.6 Pericytes originate from PDGFR-ß-positive progenitors in arterial walls and migrate along capillary endothelial sprouts that express PDGF-B. PDGF-B-deficient mice lack pericytes, indicating that pericytes require PDGF-B for their development,6 which is consistent with our observation that increased production of PDGF-B in the retina results in excessive production of pericytes.
Other in vitro26,35 and in vivo7 studies also suggest that endothelial cell-derived PDGF-B constitutes an important paracrine signal that directs pericyte recruitment along retinal microvessels. Ectopic exogenous PDGF-B,7 just like ectopic endogenous PDGF-B (this study), disrupts retinal vascular development, indicating that the temporal and spatial patterning of the paracrine signal is critical.
In addition to providing data that support a critical role for PDGF-B as an important regulator of pericyte migration and proliferation in the retina, rho/PDGFB mice provide a model of spontaneous traction retinal detachment. Traction retinal detachment is a major cause of visual loss associated with both neovascular and non-neovascular proliferative retinopathies, and expression of PDGFs is increased in each of these disease processes.5,9 Examples of the former are ischemic retinopathies, such as proliferative diabetic retinopathy and retinopathy of prematurity, and an example of the latter is proliferative vitreoretinopathy, the most common cause of failure of retinal reattachment surgery.8 Oxygen-induced ischemic retinopathy in mice or rats mimics characteristics of the early stages of human ischemic retinopathies, such as retinal neovascularization, but unlike human diseases, there is no progression to traction retinal detachment. Therefore, that model cannot be used to study the pathogenesis and treatment of traction retinal detachment, which is the blinding component of human ischemic retinopathies.
Injection of heterologous or autologous cells into the vitreous cavity of rabbits results in traction retinal detachment.36 This model and models that involve wounding the retina have been used extensively to investigate the pathogenesis and treatment of traction retinal detachments.37-42 But they involve introduction of exogenous cells and/or trauma, which are potentially important differences that raise concerns regarding the relevance of these models to spontaneous traction retinal detachments that occur in human proliferative retinopathies, such as diabetic retinopathy and proliferative vitreoretinopathy.10 The increase in expression of PDGF-B and the spontaneous occurrence of traction retinal detachment followed by the proliferation of retinal pigmented epithelial cells and retinal glia on and beneath the retina in rho/PDGFB mice are important features of traction retinal detachments in humans with these retinopathies. Therefore, rho/PDGFB mice provide a valuable new tool for the study of traction retinal detachment. Although both PDGF-A and PDGF-B are increased in proliferative retinopathies, comparison of rho/PDGF-A and rho/PDGF-B mice suggest that PDGF-B is likely to be more important in generation of the detachments. Future studies will explore in detail the cellular and molecular mechanisms responsible for the detachments and will investigate ways to prevent them. Such studies could help to provide new treatments for patients with proliferative retinopathies.
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Footnotes |
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Supported by PHS grants EY05951, EY12609, EY10017, EY09769, and core grant P30EY1765 from the National Eye Institute, a Juvenile Diabetes Foundation fellowship grant (to N. O.), a Lew R. Wasserman Merit Award (to P. A. C.), a career development award (to D. J. Z.), and an unrestricted grant from Research to Prevent Blindness, Inc., the Rebecca P. Moon, Charles M. Moon, Jr., and Dr. P. Thomas Manchester Research Fund, a grant from the Association for Retinopathy of Prematurity and Related Diseases, a grant from Mrs. Harry J. Duffey, and a grant from Dr. and Mrs. William Lake. P. A. C. is the George S. and Dolores Dore Professor of Ophthalmology.
M. S. S. and N. O. contributed equally to this manuscript.
Dr. Seo’s current address: Chonnam National University Medical School and Hospital, Kwangju, Korea.
Accepted for publication May 19, 2000.
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