Abstract
purpose. The action of growth factors is thought to make a substantial
contribution to the events leading to proliferative vitreoretinopathy
(PVR). In this study, the importance of platelet-derived growth factor
(PDGF) was tested in a rabbit model of PVR.
methods. The approach was to compare the extent of PVR induced by cells
that do or do not express the receptors for PDGF and therefore differ
in their ability to respond to PDGF.
results. Mouse embryo fibroblasts derived from PDGF receptor knock-out embryos
that do not express either of the two PDGF receptors induced PVR poorly
when injected into the eyes of rabbits that had previously undergone
gas vitrectomy. Re-expression of the PDGF β receptor in these cells
did not improve the ability of the cells to cause PVR. In contrast,
injection of cells expressing the PDGF α receptor resulted in stage 3
or higher PVR in 8 of 10 animals.
conclusions. These findings show that PDGF makes an important contribution to the
development of PVR in this animal model. Furthermore, there is a marked
difference between the two receptors for PDGF, and it is the PDGF α
receptor that is capable of driving events that lead to
PVR.
Proliferative vitreoretinopathy (PVR) occurs after rhegmatogenous
retinal detachment and is characterized by the growth and contraction
of cellular membranes within the vitreous cavity and on both surfaces
of the retina. Contraction of the epiretinal membrane (ERM) leads to
tractional retinal detachments or can reopen previously treated retinal
breaks. PVR occurs in up to 10% of all cases of rhegmatogenous retinal
detachment and remains a major obstacle to improving the long-term
outcome of retinal detachment surgery.
1 2 3 4 5
In an attempt to understand the basis of this disease, a number of
investigators have focused on the composition of the ERM. It is largely
fibrous material, containing retinal pigment epithelial (RPE) cells
and, to a lesser extent, glial cells.
6 7 8 There are
numerous growth factors associated with the ERM and/or secreted by the
RPE cells.
9 10 11 Expression of platelet-derived growth
factor (PDGF)-AA, one of the three isoforms of PDGF, is increased in
RPE cells within the ERM of human patients.
12 Animal
models also show increased PDGF-AA expression in RPE cells after either
retinal detachment or laser damage.
13 14 Because RPE cells
also express the receptors for PDGF,
9 it is possible that
retinal insult triggers a PDGF-mediated autocrine loop in RPE cells.
This idea is supported by the observation that the growth of cultured
human RPE cells can be partially blocked by neutralizing antibodies to
PDGF.
13
PDGF is a dimeric polypeptide that occurs in several different forms:
PDGF-BB, PDGF-AB, and PDGF-AA.
15 16 The different isoforms
of PDGF appear to have distinct functions, because knocking out each of
the genes for PDGF leads to distinct phenotypes.
17 18 This
is, at least in part, because there are two different PDGF receptor
(PDGFR) subunits, and the composition of the receptor (which is a
ligand-inducible dimer of two subunits) is determined by the isoform of
PDGF. For instance, PDGF-BB is the universal ligand, and it assemblesαα
and ββ homodimers and αβ heterodimers, whereas PDGF-AA
activates only αα homodimers. Once activated, the PDGFR initiates
signal relay cascades that drive biologic responses, such as chemotaxis
and proliferation. Although these data strongly implicate PDGF (and in
particular PDGF-AA) as a contributor to the pathologic events leading
to PVR, it is likely that PDGF is not the only growth factor involved.
We have recently found that the receptor for hepatocyte growth factor
is expressed by RPE cells and that it stimulates migration of cultured
RPE cells. Furthermore, ERMs from patients with PVR are strongly
positive for the hepatocyte growth factor receptor.
19 In
addition, others have found that RPE cells secrete vascular endothelial
growth factor (VEGF), express receptors for VEGF, and respond
mitogenically and chemotactically to VEGF.
10 11 20 Consequently, VEGF may also play a role in PVR. Together, these
findings have lead to the hypothesis that formation of the ERM is at
least in part driven by growth factor-mediated proliferation and
chemotaxis of RPE cells.
1 2 5 21 Although many growth
factors have been implicated in PVR, the relative contribution of even
the best candidate (PDGF) has not been tested.
In this study, we directly tested the hypothesis that PDGF is important
for PVR in a rabbit model of the disease. This was accomplished by
using a novel approach of comparing the PVR potential of cells that
differed in the ability to respond to PDGF. Our findings strongly
implicate the αPDGFR in our animal model of PVR. Importantly, theα
PDGFR is selectively activated by PDGF-AA, the isoform that has been
strongly tied to PVR in humans. Finally, our data demonstrate that the
contribution of the receptor for a single growth factor can
dramatically influence the incidence of disease.
Cells were plated in 24-well dishes at 6 ×
104 cells per well in DMEM plus 10% fetal bovine
serum (FBS) and incubated at 37°C for 24 hours. Cells were washed
twice with phosphate-buffered saline (PBS) and then incubated in DMEM
plus 2 mg/ml BSA for 48 hours. Buffer (10 mM acetic acid and 2 mg/ml
BSA), PDGF AA (50 ng/ml), PDGF-BB (50 ng/ml), or FBS (10%) was added
for 18 hours, and then the cells were pulsed with[ 3H]thymidine (0.8 μCi/ml) for 4 hours. Cells were
washed with ice-cold PBS, then with 5% trichloroacetic acid and lysed
in 0.25N NaOH. Lysates were transferred into scintillation vials
containing 50 μl of 6 N HCl and 4 ml of scintillation cocktail (ICN,
Costa Mesa, CA). Incorporated radioactivity was determined by
scintillation counting. Each experimental condition was assayed in
triplicate. Two independent experiments were performed and produced
similar results.
In this study we investigated the importance of PDGF in an animal
model of PVR by comparing the PVR-inducing potential of cells that
differ in their responsiveness to PDGF. We found that expression of the
PDGFRs greatly enhanced the cells’ intrinsic ability to induce the
disease. In addition, our findings strongly implicate αPDGFR in PVR.
Because this receptor is selectively activated by PDGF-AA, our data
support and extend the data of others collected from animal models or
humans, that PDGF-AA may play an important role in PVR.
We injected mouse cells into rabbits, and consequently, the injected
cells could have been rejected. Throughout the course of the study the
vitreous of the eyes that were injected did not become cloudy or turbid
(Anthony Andrews, unpublished observations, 1998), consistent
with the idea the eyes were not becoming uveitic. A possible
explanation is that the eye is an immune-privileged site and can
tolerate foreign cells. Importantly, other investigators have also used
heterologous cells to study PVR in rabbits without any apparent
immunologic complications.
32
Cells that did not express PDGFR were only marginally able to induce
PVR
(Fig. 3) . This was an unexpected result, because PVR can be induced
by the injection of a variety of cell types, including several
different types of fibroblasts.
32 42 The cells used in
these studies were normal mouse embryo fibroblasts (with the exception
of the absence of PDGFRs), which are responsive to numerous growth
factors. For instance, they grew well in tissue culture medium
supplemented with 10% FBS, in which lysophosphatidic acid is the major
mitogen. In addition, expression of either or both of the PDGFRs did
not markedly improve the growth of these cells in serum, indicating
that PDGF is not the only mitogen for these cells. The inability to
induce PVR was most surprising when the F cells were coinjected with
PRP, a rich source of numerous growth factors. Thus, although many
growth factors have been implicated in PVR, the results of our studies
indirectly indicate that PDGF plays a particularly important role, at
least in this animal model of the disease.
Our data not only showed that PDGF is important in PVR but also began
to identify the relative contribution of each of the PDGFR subunits. In
the presence of PRP, the Fα cells induced PVR much better than cells
without any PDGFRs
(Fig. 3B) . This indicates that presence of theα
PDGFR greatly increased the likelihood of PVR. There was a
particularly wide range in stages of PVR in the group of animals
injected with the Fα cells, but no PRP
(Fig. 3A) . Because ocular
injury stimulates expression of PDGF,
9 a possible
explanation is that the surgical procedures induced expression of PDGF
to various degrees in individual rabbits within the group. Thus when
animals were injected with cells that have a high PVR potential (Fα
cells), the amount of PDGF (or other growth factors) could be the
determining factor in the severity of the disease.
In contrast to the Fα cells, the cells expressing the βPDGFR
largely did not induce the severe stages of PVR in the presence or
absence of PRP. The inability of the βPDGFR to mediate this response
is not because this receptor was nonfunctional
(Fig. 2) . The βPDGFR
initiated signal relay cascades and promoted cell cycle progression at
least as well as the αPDGFR
(Fig. 2) . Furthermore, it is unlikely
that the βPDGFR did not promote PVR because of the absence of
PDGF-BB, because this form of PDGF is readily found in the platelets of
nonprimates
44 and consequently should have been present in
the PRP.
The data showing cells coexpressing both of the PDGFRs are the most
difficult to interpret for two reasons. First, three types of receptor
dimers can form, αα and ββ homodimers and αβ heterodimers,
and it is therefore not possible to know which type of receptors is
responsible for mediating an effect. This caveat highlights the utility
of having matched sets of cells that individually express each of the
PDGFRs, in which only one type of receptor dimer is possible. The
second problem is that PRP seemed to suppress the ability of the
Fαβ cells to drive the most severe forms of PVR
(Fig. 3) , although
the difference between these two groups did not reach statistical
significance. One explanation is that the PRP contains high levels of
PDGF-BB, leading to activation of all possible types of PDGFRs and that
one or more of these PDGFRs prevent PVR. Given that the βPDGFR did
not induce disease, we speculate that the βPDGFR (ββ homodimers)
or αβ heterodimers suppress PVR that is induced through αPDGFR.
It is also likely that other variables make an important contribution
to the overall effect. For instance, transforming growth factor β,
which is present in the vitreous and retina,
45 has been
shown to induce secretion of PDGF-AA in fibroblasts.
46 Furthermore, it is plausible that at least some of the other growth
factors that have been implicated in PVR are also making a
contribution. Additional experimentation will be required to
investigate these possibilities further.
The idea that the PDGFRs make unequal contributions to PVR is
surprising, because the α and βPDGFRs are able to initiate cell
signaling, cell movement, and cell proliferation, responses that are
intrinsic to PVR. However, mice nullizygous for each of the receptors
display distinct abnormalities, suggesting that the α and βPDGFRs
play distinct roles during embryogenesis.
16 Furthermore,
although PDGF is implicated in a variety of diseases, the relative
contribution of the two receptors is nonidentical.
16 Thus,
the two PDGFRs appear to have distinct roles in both the normal and
pathologic processes.
It is likely that at least part of the reason why the two receptors
drive distinct biologic responses is because they initiate nonidentical
signal relay cascades. Indeed, although there are many similarities in
the signaling events initiated by the two PDGFRs, a number of
fundamental differences are beginning to emerge.
47 48 49 The
availability of a panel of αPDGFR mutants that are defective in
initiating one or more signal relay cascades will enable us to identify
the signaling enzymes that contributed to the progression and
establishment of PVR in this animal model. This information may provide
new targets for therapeutic intervention as well as prevention of PVR.
Finally, an important area for future investigation is to relate our
results in this animal model to the disease in humans.
Supported by the SERI Ocular Gene Therapy Program and Grant EY00327 from the National Institutes of Health.
Submitted for publication February 5, 1999; revised April 16, 1999; accepted June 3, 1999.
Commercial relationships policy: N.
Corresponding author: Andrius Kazlauskas, Schepens Eye Research
Institute, Harvard Medical School, 20 Staniford Street, Boston MA
02114. E-mail:
[email protected]
The authors thank Yasushi Ikuno, Kameran Lashkari, and Stephan
Rosenkranz for critically reading the manuscript and Ann Elsner for
help with statistical analysis.
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