October 1999
Volume 40, Issue 11
Free
Retina  |   October 1999
Platelet-Derived Growth Factor Plays a Key Role in Proliferative Vitreoretinopathy
Author Affiliations
  • Anthony Andrews
    From Retina Associates, Boston, Massachusetts; and
  • Egle Balciunaite
    Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts; and the
  • Fee Lai Leong
    Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts; and the
  • Michelle Tallquist
    Fred Hutchinson Cancer Research Center, Seattle, Washington.
  • Philippe Soriano
    Fred Hutchinson Cancer Research Center, Seattle, Washington.
  • Miguel Refojo
    Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts; and the
  • Andrius Kazlauskas
    Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts; and the
Investigative Ophthalmology & Visual Science October 1999, Vol.40, 2683-2689. doi:
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      Anthony Andrews, Egle Balciunaite, Fee Lai Leong, Michelle Tallquist, Philippe Soriano, Miguel Refojo, Andrius Kazlauskas; Platelet-Derived Growth Factor Plays a Key Role in Proliferative Vitreoretinopathy. Invest. Ophthalmol. Vis. Sci. 1999;40(11):2683-2689.

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      © ARVO (1962-2015); The Authors (2016-present)

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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. 
Materials and Methods
Expression of PDGFR
The human α and βPDGFR cDNA were subcloned into the pLXSHD and pLXSH retroviral vectors, 22 respectively, and the resultant plasmids were introduced into PA137 cells, an amphotropic virus-producing cell line, as previously described. 23 The virus was used to infect F cells, mouse embryo fibroblasts that are nullizygous for both of the PDGFRs. These cells were derived as follows: E 9.5 embryos were recovered from timed matings of PDGFR alpha+/−, PDGFR beta+/− X PDGFR alpha+/−, and PDGFR beta+/− mice, 24 25 which were on a hybrid (C57BL6x129sv) background. Embryos were dissociated for 5 minutes with 0.25% trypsin and 1 mM EDTA at 37°C. The cells were then plated on 30-mm gelatinized dishes and immortalized by infection with retroviral vector harboring the simian virus 40 large T antigen. 26 Cells were passaged at a 1:10 dilution every 3 days. After infection of the cells with the PDGFR viruses, the cells were selected in the presence of 5 mM histidinol (αPDGFR) or 200μ g/ml hygromycin (βPDGFR). Coexpression of both receptors was achieved by infecting the αPDGFR cells (Fα) with the βPDGFR virus, and the cells were grown in the presence of both histidinol and hygromycin. 
Western Blot Analysis
To determine the level to which the PDGFRs were expressed, cells were grown to confluence, washed twice with 20 mM Hepes (pH =7.4) and 150 mM NaCl (HS) and lysed in EB (10 mM Tris-HCl [pH 7.4], 5 mM EDTA, 50 mM NaCl, 50 mM NaF, 1% Triton X-100, 20 μg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride). The lysates were centrifuged for 15 minutes at 13,000 rpm (refrigerated microfuge; Savant Instruments, Farmingdale, NY), the protein content of the clarified lysates was determined with the BCA protein assay (Pierce, Rockford, IL), and then 30 μg protein was resolved by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred onto membranes (Immobilon; Amersham, Arlington Heights, IL), and the membrane was subjected to western blot analysis using anti-αPDGFR (27P/80.8) or anti-βPDGFR (30A) antibodies, as previously described. 27 28  
To measure PDGF-dependent Erk activation, cells were grown to 80% to 90% confluence, starved in Dulbecco’s modified Eagle’s medium (DMEM) plus 0.1% calf serum for 24 hours and then stimulated with 40 ng/ml of PDGF-AA or PDGF-BB for 5 minutes. The cells were washed with HS plus 2 mM sodium orthovanadate and lysed in EB plus 2 mM sodium orthovanadate. The clarified lysates were subjected to BCA protein assay, and 30 μg protein was resolved on 7.5% SDS-PAGE and transferred onto membranes. Membranes were probed with primary anti phosphoErk antibodies (1:500 dilution; New England Biolabs, Beverly, MA) and then with a horseradish peroxidase–conjugated goat anti-mouse secondary antibody (1:5000; Amersham). The signal was detected by enhanced chemiluminescence (Amersham). Western blot analysis with antibodies against the GTPase activating protein of Ras (RasGAP) was performed as previously described. 29  
[3H]Thymidine Uptake
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. 
Rabbit Surgery
Eighty pigmented rabbits in eight groups of 10 were used. The groups consisted of animals that were injected with either of the four cell lines (F, Fα, Fβ, or Fαβ), in the presence or absence of platelet-rich plasma (PRP). All rabbits were anesthetized with an intramuscular injection of 0.5 ml chloropromazine HCl (25 mg/ml) and 1 ml ketamine HCl (100 mg/ml) per kilogram. In all cases, only one of the eyes was subjected to treatment. Gas compression of the vitreous was accomplished according to standard procedures. 30 .31 Briefly, 0.4 ml perfluoropropane (C3F8) gas was injected into the vitreous through a 30-gauge needle 4 mm posterior to the limbus. A paracentesis created by a 30-gauge needle at the limbus was used to remove 0.2 ml of vitreous to restore normal eye pressure. The gas was removed on the third day after the injection. To prepare the cells for injection, dishes of confluent cells were trypsinized, washed, and resuspended in DMEM at a final concentration of 1 million cells/ml. Immediately after the removal of the C3F8 gas, 100,000 cells in 0.1 ml DMEM were slowly injected into the vitreous cavity through a 30-gauge needle, 4 mm posterior to the limbus. PRP was prepared by collecting venous rabbit blood in the presence of 3.8% sodium citrate, the whole blood was centrifuged, and the supernatant was the PRP. In experiments in which PRP was used, 0.1 ml of the PRP was injected by a second, independent injection. The fundus was checked after the injection for iatrogenic tears or retinal detachment. One animal was excluded from the study because of the occurrence of retinal detachment after an iatrogenic retinal tear. Rabbit eyes with small, limited retinal tears that did not result in retinal detachment by the end of the study period remained in the study. In a separate study we found that PVR did not develop in animals that received such a tear but had not been injected with cells (Anthony Andrews and Kameran Lashkari, unpublished observations, 1998). 
Examination of the Rabbits
The rabbits were examined by slit lamp biomicroscopy and indirect ophthalmoscope with a +30-D fundus lens through a dilated pupil (1% cyclopentolate HCl eye drops and 2.5% phenylephrine HCl eye drops, 0.05 ml of each). Each animal was examined at the outset of the experiment to rule out the presence of any pre-existing anterior and posterior segment ocular abnormalities. All procedures were performed under aseptic conditions and pursuant to the regulations of the ARVO Statement for the use of Animals in Ophthalmic and Vision Research. The rabbits were examined by the same examiner on days 1, 3, 5, and 7 and weekly thereafter using the indirect ophthalmoscope. Clinical observations were recorded according to the Fastenberg classification, 32 33 and sketches were made. The animals were killed on day 28. Eyes that received a grading of Fastenberg stage 3 or higher were enucleated, bissected, and examined in gross. Selected specimens, including the one with the iatrogenic tear, were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined by light microscopy. 
Statistical Analysis
To determine whether the differences among groups of rabbits were statistically significant we performed the Mann–Whitney test for nonparametric ordinal data. The responses of rabbits injected with the F cells were compared with the responses of rabbits injected with cells that were expressing the PDGFRs. P < 0.05 was considered to show a statistically significant difference. 
Results
Generation and Characterization of Cell Lines
To assess the role of PDGF in PVR we used cell lines that differed in their ability to respond to PDGF. To generate such cell lines we started with mouse embryo fibroblasts derived from animals that were nullizygous for all PDGFRs (F cells). Each of the PDGFRs was re-expressed in these cells by infecting them with a replication-incompetent retrovirus that harbors one of the human PDGFRs. Cells expressing both of the receptors were made by infecting the αPDGFR-expressing cells (Fα) with the βPDGFR virus. The infected cells were selected in drug-containing medium, and mass populations of cells were analyzed for expression of the introduced receptor. Confluent cultures of cells were lysed, the insoluble material was removed by centrifugation, and the resultant lysates were subjected to western blot analysis using antibodies that specifically recognize either of the two PDGFRs. In these studies the parental F cells were included as a negative control. Two positive control cell lines were also included. One was rabbit conjunctival primary fibroblasts, which are often used in the PVR rabbit model. 33 34 35 36 The second was PhαWT cells, which are a mouse embryo fibroblast cell line that expresses each of the PDGFRs at levels that are usually found in fibroblasts (approximately 100,000 receptors/cell). 37 As shown in Figure 1 A, the αPDGFR was readily detected in the Fα cells as well as the cells infected with both of the PDGFR viruses (Fαβ). The level of the αPDGFR in the Fα was comparable to that seen in conjunctival fibroblasts, and approximately three to four times less than the level of the αPDGFR in the PhαWT. The αPDGFR in the Fαβ cells was roughly two times higher than in Fα cells. In contrast, no αPDGFR was detected in either the parental F cells, or in the cells infected with the βPDGFR. The cells that had been infected with the βPDGFR virus expressed the βPDGFR, and the levels were comparable to the conjunctival fibroblasts and PhαWT cells (Fig. 1B) . Thus, both of the PDGFRs were expressed to physiologically relevant levels, and periodic assessment of the cells indicated that expression was stable for at least 6 months. 
We next characterized the ability of the introduced PDGFRs to trigger signal relay events as well as biologic responses. The microtubule–associated protein (MAP) kinase cascade is a well-characterized signal relay pathway initiated by PDGF, and we compared the panel of F cells for their ability to activate Erk, which is one of the MAP kinase family members. 38 Cells were grown to near confluence, then arrested by serum deprivation, and then left resting or exposed to 40 ng/ml PDGF-AA or PDGF-BB. PDGF-AA selectively activates the αPDGFR, whereas PDGF-BB is the universal ligand, and it activates all PDGFRs. After stimulation, the cells were lysed, and the clarified lysates were subjected to western blot analysis using a phospho-specific Erk antibody. As shown in Figure 2 A, exposure of F cells to either form of PDGF did not activate Erk, because these cells have no PDGFRs (Fig. 1) . Engagement of either theα or β PDGFR resulted in activation of Erk, and the response was consistently slightly stronger in the Fβ cells. Simultaneous activation of both of the receptors, by stimulating the Fαβ cells with PDGF-BB, did not lead to much more of a response. The lower panel of Figure 2A is a western blot of an unrelated protein in the cell lysates and is included to verify that there were similar amounts of cell lysate in all the samples. Additional studies revealed that exposure to PDGF stimulated comparable levels of tyrosine phosphorylation of the introduced receptors (data not shown). These studies indicate that the introduced receptors were capable of triggering characteristic biochemical events such as receptor autophosphorylation and coupling to the Erk pathway. 
To test whether the receptors are able to drive biologic responses, we determined whether they could initiate PDGF-dependent DNA synthesis. Cells were plated, arrested by serum deprivation, exposed to 50 ng/ml PDGF-AA or PDGF-BB for 18 hours, and then pulsed with[ 3H]thymidine to measure DNA synthesis. The F cells did not respond to either form of PDGF; however, 10% FBS initiated a readily detectable response (Fig. 2B) . Activation of αPDGFR was able to initiate entry of cells into the S phase, and the response was approximately 45% to 50% of the response seen with 10% FBS. Theβ PDGFR also stimulated DNA synthesis and was routinely somewhat better than the αPDGFR. This observation is consistent with the work of other groups that reported that the βPDGFR is more potent than theα PDGFR at driving cell cycle progression in fibroblasts. 39 40 41 Simultaneous activation of both the receptors (by PDGF-BB in Fαβ cells) resulted in a response comparable to that seen when only the βPDGFR was activated (exposure of Fβ cells to PDGF-BB). Taken together, the data in Figure 2 show that the βPDGFR was slightly more potent than the αPDGFR and that both of the receptors coupled to signal relay pathways and initiate biologic responses. In addition, simultaneous activation of both of the receptors did not dramatically change the response, compared with activation of either receptor alone. 
Comparison of Cell Lines for Their Ability to Induce PVR
To test the PVR induced by the various cell lines, we used a well-established rabbit model. Rabbits were first subjected to gas vitrectomy, then either F, Fα, Fβ, or Fαβ cells were injected, and the eyes were monitored for development of membranes and retinal detachment for 28 days. The data presented in Figure 3 are the results at day 28, and although PVR at the earlier time points was less severe, there were no additional trends between the cell lines (data not shown). All PVR in the clinical Fastenberg classifications of stage 3 or higher was confirmed on gross and light microscopic examination of the sectioned specimens. Light microscopy confirmed the presence of retinal membranes and retinal detachment in the embedded specimens. 
We found that cells without any PDGFRs (F cells) were not particularly effective in inducing PVR, with only 2 of 10 animals showing stage 3 (Fig. 3A) . Expression of the αPDGFR (Fα cells) resulted in a modest trend toward the more severe stages of PVR; however, this increase was not statistically significant (P = 0.104). Cells expressing the βPDGFR (Fβ cells) did not induce more severe PVR than the parental cells. In contrast, stage 3 or higher PVR developed in 9 of 10 rabbits when cells coexpressing the two PDGFRs (Fαβ cells) were injected. These findings indicate that expression of both of the PDGFRs greatly increased the incidence of PVR. Because there was not an exogenous source of PDGF in these experimental conditions, it appears that there is enough PDGF in the rabbit eye to activate the injected cells. 
We were somewhat surprised that the F cells were unable to induce PVR. With the exception of the PDGF receptors, these cells should express all other cell surface receptors found on fibroblasts, and fibroblasts efficiently induce PVR in this animal model. 32 35 42 Therefore, either the PDGFRs are critical for PVR, or there is not a sufficient concentration of growth factors in the injected eye to cause disease. To distinguish between these two possibilities, we altered the experimental protocol by injecting PRP together with the cells. Previous investigations have found that injection of PRP greatly accelerates the progression of PVR. 35 42 This is presumably because of the many factors within the PRP, one of which is PDGF. 43 The PRP did not have a pronounced effect on the onset of the disease (data not shown). However, it greatly potentiated the ability of the Fα cells to induce PVR: Eight of 10 rabbits had stage 3 or higher (Fig. 3B ; the difference between F and Fα was statistically significant). In contrast, PRP did not improve the ability of the Fβ cells to cause disease. The Fαβ cells were also able to induce PVR under these conditions. The findings indicate that the signal sent by the αPDGFR was critical for inducing PVR in this animal model. 
To document and verify PVR, the rabbit eyes were photographed, and then the animals were killed and the eyes enucleated and examined by gross and light microscopy. Figure 4 A shows a normal retina (left) and mild fibrous proliferation on the surface of the retina at day 28 in an animal that was injected with Fβ cells and no PRP. PVR in this animal was classified as Fastenberg stage 1. On the right is the eye of a rabbit that was injected with Fα cells together with PRP. There was extensive fibrous proliferation and consequent total retinal detachment. PVR in this animal was classified as Fastenberg stage 5. During the entire course of the study, the appearance of the membranes did not reflect the type of cells that were injected. We also examined the membranes histologically and found that the membranes were attached to the surface of the retina and that they were composed mostly of fibrous tissue, with a lesser cellular component. In addition, the retina appeared intact, and no cellular invasion was seen (Fig. 4B)
Discussion
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. 
 
Figure 1.
 
Expression of PDGFR. Confluent cultures of parental F cells (F), cells expressing αPDGFR (Fα), βPDGFR (Fβ), or both (Fαβ) were lysed, the insoluble fraction was removed by centrifugation, and 30μ g of the resultant lysate was separated by SDS-PAGE, then transferred onto membranes and subjected to western blot analysis using antisera specific for the PDGF α subunit (A) and for the PDGF β subunit (B). PhαWT are mouse embryo fibroblasts that express approximately 105 receptors/cell for each of the two PDGFRs 28 ; also included in this panel were primary rabbit conjunctival cells (C). The βPDGFR was consistently a smaller size in conjunctival fibroblasts. This may relate to the extent of glycosylation of the receptor, which is unequal in different cell types. 50
Figure 1.
 
Expression of PDGFR. Confluent cultures of parental F cells (F), cells expressing αPDGFR (Fα), βPDGFR (Fβ), or both (Fαβ) were lysed, the insoluble fraction was removed by centrifugation, and 30μ g of the resultant lysate was separated by SDS-PAGE, then transferred onto membranes and subjected to western blot analysis using antisera specific for the PDGF α subunit (A) and for the PDGF β subunit (B). PhαWT are mouse embryo fibroblasts that express approximately 105 receptors/cell for each of the two PDGFRs 28 ; also included in this panel were primary rabbit conjunctival cells (C). The βPDGFR was consistently a smaller size in conjunctival fibroblasts. This may relate to the extent of glycosylation of the receptor, which is unequal in different cell types. 50
Figure 2.
 
Responsiveness of cell lines to PDGF. (A) Confluent, quiescent cultures of F, Fα, Fβ, and Fαβ cell lines were left resting (−) or treated for 5 minutes with either 40 ng/ml PDGF AA or PDGF-BB (+). The cells were lysed, and 30 μg of clarified total cell lysate was separated by SDS-PAGE and transferred onto membranes. The blot was cut in half, and the bottom and top portions were subjected to western blot analysis using anti-phosphoErk and anti-RasGAP antibodies, respectively. (B) Proliferating cultures of F, Fα, Fβ, and Fαβ cells were plated at subconfluent cell density (50%–60%), serum starved, and then exposed to buffer, 50 ng/ml PDGF AA, 50 ng/ml PDGF-BB, or 10% FBS. After 18 hours, cells were pulsed for 4 hours with [3H]thymidine, and the amount of incorporated radioactivity was measured by scintillation counting. Each bar represents the mean ± SD of three determinations. Where no error is shown, the error is within the symbol. Similar results were obtained in two independent experiments.
Figure 2.
 
Responsiveness of cell lines to PDGF. (A) Confluent, quiescent cultures of F, Fα, Fβ, and Fαβ cell lines were left resting (−) or treated for 5 minutes with either 40 ng/ml PDGF AA or PDGF-BB (+). The cells were lysed, and 30 μg of clarified total cell lysate was separated by SDS-PAGE and transferred onto membranes. The blot was cut in half, and the bottom and top portions were subjected to western blot analysis using anti-phosphoErk and anti-RasGAP antibodies, respectively. (B) Proliferating cultures of F, Fα, Fβ, and Fαβ cells were plated at subconfluent cell density (50%–60%), serum starved, and then exposed to buffer, 50 ng/ml PDGF AA, 50 ng/ml PDGF-BB, or 10% FBS. After 18 hours, cells were pulsed for 4 hours with [3H]thymidine, and the amount of incorporated radioactivity was measured by scintillation counting. Each bar represents the mean ± SD of three determinations. Where no error is shown, the error is within the symbol. Similar results were obtained in two independent experiments.
Figure 3.
 
The PVR potential of the various cell lines. Rabbits were subjected to gas compression vitrectomy, and then cells expressing the PDGFRs (as described in the legend of Fig. 1 ) were injected in the absence (A) or presence (B) of PRP. The animals were examined by the same examiner with an indirect ophthalmoscope at days 1, 3, 5, 7, 14, 21, and 28 after the injection. The data presented are the findings at day 28. Each point in the figure is the Fastenberg classification of PVR for an individual rabbit. All the data were collected as integers but were made either slightly smaller or larger to make it possible to see each point in the figure. The asterisks indicate a statistically significant difference (between F cells and the other groups of cells), and P is shown above the panels; the horizontal bars indicate the mean of the data set.
Figure 3.
 
The PVR potential of the various cell lines. Rabbits were subjected to gas compression vitrectomy, and then cells expressing the PDGFRs (as described in the legend of Fig. 1 ) were injected in the absence (A) or presence (B) of PRP. The animals were examined by the same examiner with an indirect ophthalmoscope at days 1, 3, 5, 7, 14, 21, and 28 after the injection. The data presented are the findings at day 28. Each point in the figure is the Fastenberg classification of PVR for an individual rabbit. All the data were collected as integers but were made either slightly smaller or larger to make it possible to see each point in the figure. The asterisks indicate a statistically significant difference (between F cells and the other groups of cells), and P is shown above the panels; the horizontal bars indicate the mean of the data set.
Figure 4.
 
Photographs of the rabbit eyes and of the epiretinal membranes. (A) Photographs of the retinas of living rabbits. Normal retina (left); fibrous proliferation on the surface of the retina, Fastenberg stage 1 (middle); fibrous proliferation centrally, with total retinal detachment, Fastenberg stage 5 (right). (B) Photographs of histologic sections stained with hematoxylin–eosin. Left, a normal rabbit retina; right, fibrous proliferation on the surface of the retina. Magnification,× 20.
Figure 4.
 
Photographs of the rabbit eyes and of the epiretinal membranes. (A) Photographs of the retinas of living rabbits. Normal retina (left); fibrous proliferation on the surface of the retina, Fastenberg stage 1 (middle); fibrous proliferation centrally, with total retinal detachment, Fastenberg stage 5 (right). (B) Photographs of histologic sections stained with hematoxylin–eosin. Left, a normal rabbit retina; right, fibrous proliferation on the surface of the retina. Magnification,× 20.
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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Figure 1.
 
Expression of PDGFR. Confluent cultures of parental F cells (F), cells expressing αPDGFR (Fα), βPDGFR (Fβ), or both (Fαβ) were lysed, the insoluble fraction was removed by centrifugation, and 30μ g of the resultant lysate was separated by SDS-PAGE, then transferred onto membranes and subjected to western blot analysis using antisera specific for the PDGF α subunit (A) and for the PDGF β subunit (B). PhαWT are mouse embryo fibroblasts that express approximately 105 receptors/cell for each of the two PDGFRs 28 ; also included in this panel were primary rabbit conjunctival cells (C). The βPDGFR was consistently a smaller size in conjunctival fibroblasts. This may relate to the extent of glycosylation of the receptor, which is unequal in different cell types. 50
Figure 1.
 
Expression of PDGFR. Confluent cultures of parental F cells (F), cells expressing αPDGFR (Fα), βPDGFR (Fβ), or both (Fαβ) were lysed, the insoluble fraction was removed by centrifugation, and 30μ g of the resultant lysate was separated by SDS-PAGE, then transferred onto membranes and subjected to western blot analysis using antisera specific for the PDGF α subunit (A) and for the PDGF β subunit (B). PhαWT are mouse embryo fibroblasts that express approximately 105 receptors/cell for each of the two PDGFRs 28 ; also included in this panel were primary rabbit conjunctival cells (C). The βPDGFR was consistently a smaller size in conjunctival fibroblasts. This may relate to the extent of glycosylation of the receptor, which is unequal in different cell types. 50
Figure 2.
 
Responsiveness of cell lines to PDGF. (A) Confluent, quiescent cultures of F, Fα, Fβ, and Fαβ cell lines were left resting (−) or treated for 5 minutes with either 40 ng/ml PDGF AA or PDGF-BB (+). The cells were lysed, and 30 μg of clarified total cell lysate was separated by SDS-PAGE and transferred onto membranes. The blot was cut in half, and the bottom and top portions were subjected to western blot analysis using anti-phosphoErk and anti-RasGAP antibodies, respectively. (B) Proliferating cultures of F, Fα, Fβ, and Fαβ cells were plated at subconfluent cell density (50%–60%), serum starved, and then exposed to buffer, 50 ng/ml PDGF AA, 50 ng/ml PDGF-BB, or 10% FBS. After 18 hours, cells were pulsed for 4 hours with [3H]thymidine, and the amount of incorporated radioactivity was measured by scintillation counting. Each bar represents the mean ± SD of three determinations. Where no error is shown, the error is within the symbol. Similar results were obtained in two independent experiments.
Figure 2.
 
Responsiveness of cell lines to PDGF. (A) Confluent, quiescent cultures of F, Fα, Fβ, and Fαβ cell lines were left resting (−) or treated for 5 minutes with either 40 ng/ml PDGF AA or PDGF-BB (+). The cells were lysed, and 30 μg of clarified total cell lysate was separated by SDS-PAGE and transferred onto membranes. The blot was cut in half, and the bottom and top portions were subjected to western blot analysis using anti-phosphoErk and anti-RasGAP antibodies, respectively. (B) Proliferating cultures of F, Fα, Fβ, and Fαβ cells were plated at subconfluent cell density (50%–60%), serum starved, and then exposed to buffer, 50 ng/ml PDGF AA, 50 ng/ml PDGF-BB, or 10% FBS. After 18 hours, cells were pulsed for 4 hours with [3H]thymidine, and the amount of incorporated radioactivity was measured by scintillation counting. Each bar represents the mean ± SD of three determinations. Where no error is shown, the error is within the symbol. Similar results were obtained in two independent experiments.
Figure 3.
 
The PVR potential of the various cell lines. Rabbits were subjected to gas compression vitrectomy, and then cells expressing the PDGFRs (as described in the legend of Fig. 1 ) were injected in the absence (A) or presence (B) of PRP. The animals were examined by the same examiner with an indirect ophthalmoscope at days 1, 3, 5, 7, 14, 21, and 28 after the injection. The data presented are the findings at day 28. Each point in the figure is the Fastenberg classification of PVR for an individual rabbit. All the data were collected as integers but were made either slightly smaller or larger to make it possible to see each point in the figure. The asterisks indicate a statistically significant difference (between F cells and the other groups of cells), and P is shown above the panels; the horizontal bars indicate the mean of the data set.
Figure 3.
 
The PVR potential of the various cell lines. Rabbits were subjected to gas compression vitrectomy, and then cells expressing the PDGFRs (as described in the legend of Fig. 1 ) were injected in the absence (A) or presence (B) of PRP. The animals were examined by the same examiner with an indirect ophthalmoscope at days 1, 3, 5, 7, 14, 21, and 28 after the injection. The data presented are the findings at day 28. Each point in the figure is the Fastenberg classification of PVR for an individual rabbit. All the data were collected as integers but were made either slightly smaller or larger to make it possible to see each point in the figure. The asterisks indicate a statistically significant difference (between F cells and the other groups of cells), and P is shown above the panels; the horizontal bars indicate the mean of the data set.
Figure 4.
 
Photographs of the rabbit eyes and of the epiretinal membranes. (A) Photographs of the retinas of living rabbits. Normal retina (left); fibrous proliferation on the surface of the retina, Fastenberg stage 1 (middle); fibrous proliferation centrally, with total retinal detachment, Fastenberg stage 5 (right). (B) Photographs of histologic sections stained with hematoxylin–eosin. Left, a normal rabbit retina; right, fibrous proliferation on the surface of the retina. Magnification,× 20.
Figure 4.
 
Photographs of the rabbit eyes and of the epiretinal membranes. (A) Photographs of the retinas of living rabbits. Normal retina (left); fibrous proliferation on the surface of the retina, Fastenberg stage 1 (middle); fibrous proliferation centrally, with total retinal detachment, Fastenberg stage 5 (right). (B) Photographs of histologic sections stained with hematoxylin–eosin. Left, a normal rabbit retina; right, fibrous proliferation on the surface of the retina. Magnification,× 20.
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