July 2002
Volume 43, Issue 7
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Retina  |   July 2002
An In Vivo Gene Therapy Approach for Experimental Proliferative Vitreoretinopathy Using the Truncated Platelet-Derived Growth Factor α Receptor
Author Affiliations
  • Yasushi Ikuno
    From the Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts.
  • Andrius Kazlauskas
    From the Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts.
Investigative Ophthalmology & Visual Science July 2002, Vol.43, 2406-2411. doi:
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      Yasushi Ikuno, Andrius Kazlauskas; An In Vivo Gene Therapy Approach for Experimental Proliferative Vitreoretinopathy Using the Truncated Platelet-Derived Growth Factor α Receptor. Invest. Ophthalmol. Vis. Sci. 2002;43(7):2406-2411.

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

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Abstract

purpose. Proliferative vitreoretinopathy (PVR) is a serious problem in vitreoretinal surgeries. A report of a previous study has indicated that platelet-derived growth factor α receptor (αPDGFR) plays an important role in a rabbit model of this disease and that a dominant negative αPDGFR potently suppresses PVR in an ex vivo setting. Herein, the effect of in vivo gene delivery of a dominant negative αPDGFR on PVR was tested in a rabbit model of the disease.

methods. The dominant negative αPDGFR (TαR) is a truncated version of the receptor, which does not have the intracellular domain. It was expressed by using a retrovirus. In vitro characterization of TαR was performed in primary cultures of rabbit conjunctival fibroblasts (RCFs). Western blot analysis was used to check the expression of TαR protein. A type I collagen gel contraction assay was performed to test the efficacy of TαR on PDGF-dependent cellular responses in vitro. The in vivo efficacy and specificity of the retrovirus was determined by injecting a green fluorescent protein (GFP) retrovirus into rabbits that had been preinjected with RCFs. The impact of the TαR retrovirus on PVR was tested by using the rabbit model in which PVR was induced by the injection of RCFs and platelet-rich plasma (PRP).

results. TαR was expressed at more that 50 times the level of endogenous αPDGFR in RCFs and severely reduced PDGF-dependent contraction of collagen gels. Intravitreal injection of the GFP retrovirus resulted in expression of GFP primarily in the injected RCFs. Whereas injection of RCFs induced complete retinal detachment in 100% of the animals, co-injection of the TαR retrovirus substantially reduced the severity and incidence of retinal detachments.

conclusions. Gene therapy with a retrovirus used to express a dominant negative αPDGFR attenuates PVR in a rabbit model of the disease. This strategy may be a new approach to preventing PVR in humans.

Proliferative vitreoretinopathy (PVR) is a serious problem in patients with retinal detachments. It is characterized by the migration and proliferation of retinal pigment epithelial (RPE) cells along with the synthesis of extracellular matrix (ECM) proteins, such as collagen or fibronectin, which organize into an epiretinal membrane. Contraction of the epiretinal membrane results in tractional retinal detachment. PVR occurs in 5% to 10% of the patients who undergo retinal reattachment surgery. Of this group, nearly 20% to 40% have recurring episodes of PVR and partial vision loss. 1 2  
Given that growth factors are capable of initiating many of the cellular events that are intrinsic to PVR, numerous investigators have studied whether growth factors are involved in PVR. The list of implicated growth factors includes transforming growth factor (TGF)-β, 3 4 5 hepatocyte growth factor (HGF), 6 7 basic fibroblast growth factor (bFGF), interleukin (IL)-6, 8 and platelet-derived growth factor (PDGF). Of these growth factors, PDGF’s role in PVR has been most studied. PDGF has been found in epiretinal membranes, and cultured RPE cells both secrete and respond to PDGF. 9 10 11 12 Furthermore, injuring the retina increases expression of PDGF in the RPE cells underlying the damaged retina. 13 14 In a rabbit model of PVR, cells that express PDGFRs are much better able to induce PVR than the corresponding PDGFR-negative cells. 15 In addition, expression of a dominant negative PDGFR in rabbit conjunctival fibroblasts (RCFs) severely reduces the PVR potential of these cells. 16 Taken together, these findings indicate that multiple growth factors may be involved in PVR and that PDGF is one of the essential contributors. 
PDGF is a potent mitogen for fibroblasts, induces DNA synthesis and chemotaxis, and sometimes acts as a survival factor. Four PDGF genes have been identified that encode PDGF-A, -B, -C, and -D. 17 18 19 20 Biologically active PDGF is either a homo- or heterodimer, and the receptor for PDGF is a homo- or heterodimer of the α and β subunits. 21 The receptor subunits differ in affinity for the ligand, and hence the composition of the dimeric receptor is in part dependent on the isoform of PDGF. For instance, PDGF-AA and -CC assemble αα homodimers, PDGF-AB promotes formation of αα homo- or αβ heterodimer, and PDGF-BB is the universal ligand that binds to any subunit combination. 22 The newest member of the PDGF family is PDGF-D, which activates ββ homodimers, and to a lesser extent αβ heterodimers. 17 19 PDGF dimerizes the PDGFR, which activates the receptor’s tyrosine kinase activity and initiates intracellular signaling that culminates in a variety of cellular responses. 23  
Gene therapy–based strategies have been tested in animal models for several ocular diseases including PVR and retinitis pigmentosa. 16 24 25 26 27 28 29 Consequently, it seems likely that these types of disease will be treated by gene therapy at some point in the future. 
In this study, we used the rabbit PVR model to evaluate an in vivo gene therapy-based prevention strategy. A retroviral delivery system selectively transduced green fluorescent protein (GFP) into rabbit conjunctival fibroblasts (RCFs) that had been injected into the vitreous of rabbits. Furthermore, in vivo delivery of retrovirus containing a dominant negative αPDGFR reduced the incidence and severity of retinal detachment. 
Materials and Methods
Cells
Primary cultures of rabbit conjunctival fibroblasts were prepared as previously described 30 and maintained in Dulbecco’s modified essential medium (DMEM; GibcoBRL, Grand Island, NY) and 10% fetal bovine serum (FBS; Gemini BioProducts, Calabasas, CA) and antibiotics. 
Contraction Assay
The contraction assay was conducted as previously described, 31 with slight modification. RCFs were suspended in 1.5 mg/mL of neutralized collagen I (Vitrogen 100; Cohesion Technology, Palo Alto, CA) at a density of 106 cells/mL and were transferred into a 24-well plate (Falcon, Franklin Lakes, NJ) that had been preincubated with phosphate-buffered saline (PBS) and 5 mg/mL bovine serum albumin (BSA) overnight. The gel was solidified by incubating at 37°C for 90 minutes, and then the gels were suspended in DMEM and 5 mg/mL BSA supplemented with buffer or the agent to be tested. The initial gel diameter was 15 mm and was remeasured after a 6-hour incubation at 37°C with 5% CO2. The extent of contraction was calculated by subtracting the diameter of the well at the end of the experiment from the initial diameter (15 mm). Each experimental condition was assayed in triplicate, and at least three independent experiments were performed. 
Retrovirus
The GFP and TαR cDNAs were subcloned into the CMMP vector. 32 This vector is a derivative of the MFG series of retroviral vectors. 33 The pMMP vector was constructed by substituting the Moloney murine leukemia virus (MMLV) long terminal repeats (LTRs) in the original MFG vector with the corresponding regions from myeloproliferative sarcoma virus (MPSV). In addition, the normal proline transfer (t)RNA binding site of MLV was replaced with a glutamine tRNA binding site. Both modifications were introduced to enhanced gene expression. The pCMMP vector was generated from pMMP by replacing the MPSV U3 region in the 5′ LTR of the pMMP with cytomegalovirus immediate early gene promoter (CMV IE). The TαR does not have the majority of the cytoplasmic domain of the receptor and retains the extracellular transmembrane and juxtamembrane domains (amino acids 1–589 of the human αPDGFR). 16 Expression of TαR in NIH 3T3 cells blocks PDGF-dependent cell cycle progression. 16 The replication incompetent GFP and TαR retroviruses were made using the 293 GPG system. 34 Fluorescence based cell sorting (FACS; BD Bioscience, Mountain View, CA) of infected cells was used to establish the titer of the GFP virus. The titer for the TαR virus was determined by staining infected cells with a monoclonal antibody to the extracellular domain of the primate αPDGFR (R&D Systems, Minneapolis, MN). The titers were 5.8 × 109 colony-forming units (CFU)/mL for GFP and 6.7 × 107 CFU/mL for TαR. Retrovirus harboring an empty expression vector (pLXSHD2) was used in some of experiments, and its titer was calculated by monitoring resistance of infected cells to growth in histidinol-containing medium. The titer of this virus was 1.3 × 107 CFU/mL. 
Western Blot Analysis
RCFs were grown to 80% confluence and then serum starved by incubating in DMEM and 0.1% FBS for 18 to 24 hours. Cells were washed twice with HS (20 mM HEPES [pH 7.4], 150 mM NaCl), and then lysed in EB (10 mM Tris-HCl [pH 7.4], 5 mM EDTA, 50 mM NaCl, 50 mM NaF, 1% Triton X-100, 0.1% BSA, 20 μg/mL aprotinin, 2 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride [PMSF]). Lysates were centrifuged for 15 minutes at 13,000g, the pellet was discarded and the soluble fraction used as the total cell lysate. The protein concentration was measured with a protein assay kit (Pierce, Rockford, IL), according to the manufacturer’s instruction. 
Total cell lysates containing 20 μg protein were resolved by 7.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. Proteins were transferred onto membranes (Immobilon; Millipore, Bedford, MA) and the membranes were blocked in a reagent containing 10 mg/mL nonfat dry milk and 0.05% Tween 20 in Western rinse solution (Blotto; Santa Cruz Biotechnology, Santa Cruz, CA). Membranes were incubated with primary antibodies for 1 hour at room temperature, and washed five times with Western Rinse solution (150 mM NaCl, 8 mM Tris-HCl [pH 7.5], 2 mM Tris base). The blots were then incubated with secondary antibody for 1 hour at room temperature and washed five times with Western rinse solution. Finally all blots were developed using ECL (Amersham Pharmacia Biotech, Piscataway, NJ). 
Antibodies
The 27P antibody is a crude polyclonal rabbit antibody raised against a glutathione S-transferase (GST) fusion protein including the human αPDGFR carboxyl terminus (amino acids 951–1089). The Ras GTP-activating protein (RasGAP) antibody is a crude rabbit antisera against the SH2-SH3-SH2 of the human RasGAP (69.3). For Western blots the following dilutions were used for each primary antibody: αPDGFR, 27P, 1:1000; 69.3, 1:4000. Secondary antibodies were horseradish peroxidase–conjugated goat anti-rabbit or anti-mouse antibodies (Amersham Pharmacia Biotech) diluted 1:5000. 
Microscopic Examination
RCFs were plated onto glass coverslips and cultured in DMEM and 10% FBS overnight. They were infected with the desired retrovirus, and, after an additional 24 hours, the cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature. Rabbit eyes were fixed with 4% paraformaldehyde overnight after removal of the anterior segment of the eye, which includes the lens, iris, and cornea. They were transferred into 4% paraformaldehyde and 30% sucrose for postfixation overnight at 4°C. Rabbit samples were embedded in OCT compound and 8-μm frozen sections were prepared. 
Fluorescent signals were observed with a fluorescence microscope (Carl Zeiss, Oberkochen, Germany). Cells were labeled with live orange dye (Cell Tracker; Molecular Probes, Eugene, OR) according to the manufacturer’s instruction. 
Rabbit Model for PVR
PVR was induced in the rabbit by the injection of RCFs and platelet-rich plasma (PRP) exactly as previously described. 30 Briefly, with the rabbit under anesthesia, gas vitrectomy was performed by injecting 0.4 mL of perfluoropropane (C3F8) into the vitreous cavity 4 mm posterior to the corneal limbus. Three days later, the rabbits were anesthetized, the pupils were dilated, and 1 × 105 RCFs in 0.1 mL DMEM was injected into the vitreous cavity, together with 0.1 mL of PRP using a syringe fitted with a 30-gauge needle. Either GFP virus (0.2 mL) or TαR virus (0.2 mL) was injected separately. Eleven rabbits underwent surgery in each group. A single investigator evaluated the retinal status in an unmasked fashion using an indirect ophthalmoscope fitted with a +30-D fundus lens at 1, 4, 7, 14, 21, and 28 days after the surgery. PVR grading was according to the method of Fastenberg et al. 35 The grading is as follows: 0, no abnormality; 1, vitreous strand; 2, traction of the retina; 3, retinal detachment involving less than two quadrants; 4, extended retinal detachment including more than two quadrants; and 5, total retinal detachment. All surgeries were performed under aseptic conditions and pursuant to the guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Only the left eye of each rabbit was used for the experiments. 
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 PVR scores of rabbits injected with the TαR virus were compared with the response of those injected with the GFP virus. For the in vitro collagen gel contraction assay, the unpaired t-test was used. In all cases, P < 0.01 was considered to be significant. 
Results
Characterization of the Retroviruses
To test whether the replication-incompetent retroviruses mediate expression of the desired proteins, we infected primary RCFs and then characterized the resultant cells. Approximately 1.0 × 105 cells were plated on glass coverslips in 12-well plates and cultured overnight in DMEM and 10% FBS. The cells were infected with 1.3 × 106 CFU of GFP virus or a control virus harboring an empty expression vector (pLXSHD2), as described in the Materials and Methods section. Thirty-six hours after the viruses were added, the cells were fixed with paraformaldehyde and examined under the fluorescence microscope and photographed. Approximately 80% of the cells infected with the GFP virus expressed GFP (Fig. 1A , top panel). In contrast, the cells infected with the empty vector virus did not show any signal (data not shown). 
We also tested whether TαR could be expressed in RCFs, by using the retroviral vector that we planned to use in the in vivo studies. We had previously found that a different retroviral vector was suitable for expression of TαR. 16 To this end, 1.0 × 105 RCFs were plated in a six-well dish, 7.9 × 105 CFU of the TαR virus or 5.8 × 105 CFU of the GFP virus was added, and the cultures were incubated overnight. The following morning, the medium was replaced with starvation medium (DMEM with 0.1% FBS) and incubated for 18 to 24 hours. The cells were lysed, and the lysates were subjected to Western blot analysis with an antibody that recognizes both the endogenous full-length and the truncated αPDGFR. As seen in Figure 1B , cells infected with the TαR virus expressed the ∼100-kDa TαR protein. Densitometric analysis indicated that TαR was at least 50 times more abundant than the 170-kDa endogenous αPDGFR. We concluded that replication-incompetent retroviruses were a viable route for expressing the GFP and TαR proteins in these cells. 
Effect of TαR on PDGF-Dependent Contraction
To test the effect of expression of TαR with this retroviral vector on PDGF-dependent cellular responses, we subjected RCFs infected with either GFP or TαR to an in vitro collagen gel contraction assay. This is one of the cellular responses thought to be a component of PVR. 2 RCFs infected with GFP or TαR were resuspended in a type I collagen gel and plated in a 24-well dish. The cultures were left unstimulated or exposed to 10% FBS or PDGF-AA (0.4–50 ng/mL) for 6 hours, and then the diameter of the collagen gels was measured. As shown in Figure 2 , both cell types responded comparably to FBS. In contrast, PDGF-AA–stimulated contraction was attenuated in the TαR-expressing cells, compared with the GFP-expressing cells. Note that these two cell types express similar levels of the endogenous αPDGFR (Fig. 1B) . We concluded that expression of TαR substantially inhibited PDGF-dependent contraction, whereas it had only a minor effect on the FBS-driven response. 
Selective Targeting of Injected Cells of the Rabbit Eyes
The data in Figures 1 and 2 indicated that TαR was capable of blocking PDGF-dependent cellular responses and that the retrovirus was an effective means to express this protein in a tissue culture setting. To test whether the retroviral vehicle would be suitable for in vivo expression of proteins, we performed the following experiments. Approximately 1 × 105 RCFs in 0.1 mL DMEM were co-injected with 0.1 mL PRP into a space within the rabbit vitreous created by a gas vitrectomy. Immediately after the injection of cells, 2.9 × 107 CFU of the GFP virus was injected in a total volume of 5 μL. The injected cells were prelabeled with a red fluorescent dye (Cell Tracker), to distinguish them from resident ocular cells. When stained with this dye, more than 99% of the cells appeared red under the fluorescent microscope (data not shown). PVR developed 7 days after the injections, the animals were killed, and histologic sections of the injected eyes were prepared. Under regular illumination, an epiretinal membrane was apparent that consisted of cells and collagenous tissue (Fig. 3A) . When illuminated with fluorescent light, the majority of the cells in the epiretinal membrane were red (Fig. 3B) , indicating that they were the injected cells. The GFP-positive green cells were also observed (Fig. 3C) , and all the green cells were also stained red (Fig. 3D) , indicating that the virus selectively infected the injected cells. This was the expected outcome, because retroviruses preferentially infect proliferating cells, 36 and the injected cells are actively cycling, whereas the resident cells are not. 37 Repeated attempts to detect resident cells (i.e., not red) that were positive for GFP were not successful. Of the red cells, approximately 50% were positive for GFP, indicating that the virus had infected roughly half of the injected cells. When a similar experiment was performed with GFP in an adenovirus, many of the resident cells were green (data not shown). These findings suggest that the retroviral route is both efficient and selective for the injected cells. Furthermore, the injected cells are the cells that we want to target, because they constitute the cellular component of the epiretinal membrane. 
Effect of TαR Virus on Experimental PVR in Rabbits
Finally, we tested the TαR retrovirus in the rabbit PVR model. RCFs and PRP were injected into rabbit eyes 3 days after the gas vitrectomy. Either 5 μL GFP virus (3.9 × 107 CFU) or 200 μL TαR virus (1.8 × 107 CFU) was injected on the same day. Thereafter, the rabbit eyes were observed up to 28 days. The status of the fundus was scored according to the Fastenberg method. 35 The PVR score was significantly lower (P < 0.01) in the eyes injected with TαR virus compared with the GFP virus at 14, 21, and 28 days after the surgery (Fig. 4) . In separate studies we have observed that RCFs infected ex vivo with an empty vector–containing virus induces stage 5 PVR by 7 days, 16 (i.e., faster than in the experiments shown in Fig. 4 ). We did not perform experiments in which rabbits were injected with buffer alone or a virus harboring an empty expression vector. Consequently, we do not know whether expression of GFP, introduction of a retrovirus, the injection itself, or some other variable delayed the full retinal detachment in the present study. Importantly, the findings in Figure 4 show that the TαR virus significantly attenuated PVR in this experimental rabbit model. There were no gross anatomic or morphologic changes in the retina of the virus-injected animals, indicating that the retrovirus did not precipitate ocular damage, which is consistent with the findings of other groups. 28  
Discussion
In our study, retroviruses were an efficient and selective way of targeting expression of exogenous genes in the cells that are involved in PVR. In addition, the TαR virus was capable of blocking PDGF-dependent contraction in collagen gels, one of the cellular events that correlates closely with PVR. 38 Finally, in vivo administration of the TαR retrovirus significantly reduced PVR in a rabbit model of the disease. 
The studies presented herein further support the idea that the PDGFR is essential to the development of PVR. We have found that expressing a dominant negative PDGFR in RCFs before they are injected into the rabbit markedly reduces the ability of these cells to induce PVR. 16 Furthermore, mouse embryo fibroblasts that do not have PDGFRs induce PVR poorly. 15 Re-expression of the αPDGFR or βPDGFR enables these cells to respond mitogenically, but only the αPDGFR substantially increases the PVR potential of the cells. 15 Our past and present findings indicate that functional PDGFRs greatly increase the PVR potential of cells. However, PDGF may not be the only growth factor required for PVR. Instead, it seems likely that at least some of the other growth factors that have been implicated in PVR also contribute to the disease. Eliminating any one of these may be sufficient to lessen the intensity and severity of PVR substantially, as we have found with PDGF. 
The cellular processes that are commonly thought to contribute to PVR include migration, proliferation, synthesis of extracellular matrix, and contraction. Evaluating the relative contribution of each of these components to the overall disease outcome will profoundly improve our understanding of how PVR is initiated and progresses. One way to begin the investigation is to inhibit each of the cellular responses selectively by using PDGFR mutants. The dominant negative PDGF receptor mutant used in these studies functions to prevent activation of the receptor at the very start of the signaling cascade, and hence eliminates all cellular responses. A second type of dominant negative PDGFR, V859M, is equally effective as TαR in blocking PDGF-dependent cell cycle progression, yet the V859M mutant is not as potent as TαR in preventing PVR. 16 It appears that responses other than cell proliferation make an important contribution. Given the huge amount of extracellular matrix in the epiretinal membrane, the ability to synthesize extracellular matrix may be a particularly important component of disease progression. We are actively investigating this possibility. 
The identification of the PDGFR as a target for PVR treatment opens up the possibility of pharmacologic intervention. A number of inhibitors have been recently developed that block activation of the PDGFR, and these may turn out to be a useful alternative or complement to gene therapy. Note that PDGF appears to be a trophic factor during retinal development and survival, 39 40 and hence it may be important to tailor pharmacologic approaches so that they are selective for the cell types involved in PVR (i.e., RPE cells, retinal glial cells, and migrated fibroblasts). 
How can the gene therapy–based approach to treat experimental PVR described in this study be applied in a clinical setting? Provided that the experimental model accurately reflects the disease in patients, we envision that gene therapy would be used as a prophylactic measure, which would be administered at the conclusion of surgery. Additional studies are needed to determine the timing of administration of this therapy to achieve the greatest impact on prevention of PVR. 
In conclusion, we have developed a gene therapy–based strategy to attenuate experimental PVR in a rabbit model of the disease. Because our long-term goal is to extend these findings to humans, we want to investigate whether blocking αPDGFR function prevents PVR in other models or species that are closer to PVR observed in clinical settings. 
 
Figure 1.
 
Retroviral expression of GFP and dominant negative PDGFα receptor (TαR) in primary rabbit conjunctival fibroblasts (RCFs). (A) GFP virus. RCFs were plated on a glass coverslip and infected with the GFP (top). Twenty-four hours after infection, the cells were fixed with 4% paraformaldehyde and viewed under fluorescent illumination. The fluorescent signal was detected in approximately 80% of cells infected with GFP. Cultures that were infected with a virus expressing an empty vector showed no detectable signal (data not shown). Magnification, ×200. (B) TαR virus. RCFs were plated and infected with either GFP or TαR virus, replated in 10-cm dishes, and grown to 80% confluence. The serum in the media was reduced to 0.1% for 18 to 24 hours, the cells were lysed, and 20 μg of cell lysate was subjected to Western blot analysis with an anti-αPDGFR or Ras-GAP (lysate control) antibody. Arrows: Full length (170-kDa) and truncated (100-kDa) receptors. Densitometric analysis revealed that the level of TαR was at least 50 times above the level of the endogenous full-length PDGFR.
Figure 1.
 
Retroviral expression of GFP and dominant negative PDGFα receptor (TαR) in primary rabbit conjunctival fibroblasts (RCFs). (A) GFP virus. RCFs were plated on a glass coverslip and infected with the GFP (top). Twenty-four hours after infection, the cells were fixed with 4% paraformaldehyde and viewed under fluorescent illumination. The fluorescent signal was detected in approximately 80% of cells infected with GFP. Cultures that were infected with a virus expressing an empty vector showed no detectable signal (data not shown). Magnification, ×200. (B) TαR virus. RCFs were plated and infected with either GFP or TαR virus, replated in 10-cm dishes, and grown to 80% confluence. The serum in the media was reduced to 0.1% for 18 to 24 hours, the cells were lysed, and 20 μg of cell lysate was subjected to Western blot analysis with an anti-αPDGFR or Ras-GAP (lysate control) antibody. Arrows: Full length (170-kDa) and truncated (100-kDa) receptors. Densitometric analysis revealed that the level of TαR was at least 50 times above the level of the endogenous full-length PDGFR.
Figure 2.
 
TαR attenuates PDGF-dependent contraction of RCFs. RCFs were infected with GFP or TαR and subjected to the collagen I gel contraction assay in the presence of buffer, 10% (vol/vol) FBS, or the indicated concentration of PDGF-AA. After 6 hours of exposure to the test substances, the extent of gel contraction was measured. Each experimental condition was assayed in triplicate and the mean ± SD is presented in the bar graph. *P < 0.05, **P < 0.01.
Figure 2.
 
TαR attenuates PDGF-dependent contraction of RCFs. RCFs were infected with GFP or TαR and subjected to the collagen I gel contraction assay in the presence of buffer, 10% (vol/vol) FBS, or the indicated concentration of PDGF-AA. After 6 hours of exposure to the test substances, the extent of gel contraction was measured. Each experimental condition was assayed in triplicate and the mean ± SD is presented in the bar graph. *P < 0.05, **P < 0.01.
Figure 3.
 
The retrovirus transduced GFP selectively into the cells of the epiretinal membrane. RCFs were prestained with red dye and injected into the rabbit’s eyes. The GFP virus was injected immediately afterward. Seven days later, the rabbit was killed, and the eye was removed, sectioned, and subjected to regular and fluorescence microscopic analysis. Epiretinal membrane under (A) regular illumination and (BD) UV illumination. (B) Injected cells (red fluorescein signal) and (C) infected cells (green signal). (D) Merge of (C) and (D). All micrographs are of approximately the same section. Magnification, ×200.
Figure 3.
 
The retrovirus transduced GFP selectively into the cells of the epiretinal membrane. RCFs were prestained with red dye and injected into the rabbit’s eyes. The GFP virus was injected immediately afterward. Seven days later, the rabbit was killed, and the eye was removed, sectioned, and subjected to regular and fluorescence microscopic analysis. Epiretinal membrane under (A) regular illumination and (BD) UV illumination. (B) Injected cells (red fluorescein signal) and (C) infected cells (green signal). (D) Merge of (C) and (D). All micrographs are of approximately the same section. Magnification, ×200.
Figure 4.
 
In vivo administration of the TαR attenuates PVR. Rabbits were subjected to gas vitrectomy and, after 3 days, cells, PRP, and virus were injected. Animals that were injected with GFP virus are designated as control, whereas the TαR group received the dominant negative PDGFR virus. The PVR was evaluated and scored on days 1, 4, 7, 14, 21, and 28 after surgery. The data for days 7 (left) and 28 (right) are presented. (•) Score for individual rabbits. The value at the bottom of each column is the percentage of animals that had retinal detachments (stage 3 or greater).
Figure 4.
 
In vivo administration of the TαR attenuates PVR. Rabbits were subjected to gas vitrectomy and, after 3 days, cells, PRP, and virus were injected. Animals that were injected with GFP virus are designated as control, whereas the TαR group received the dominant negative PDGFR virus. The PVR was evaluated and scored on days 1, 4, 7, 14, 21, and 28 after surgery. The data for days 7 (left) and 28 (right) are presented. (•) Score for individual rabbits. The value at the bottom of each column is the percentage of animals that had retinal detachments (stage 3 or greater).
The authors thank Tadashi Takahashi of the Kazlauskas laboratory for critical input and Richard Mulligan and Jeng-Shin Lee of the Harvard Gene Therapy Initiative for providing the GFP and TαR retroviruses. 
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Figure 1.
 
Retroviral expression of GFP and dominant negative PDGFα receptor (TαR) in primary rabbit conjunctival fibroblasts (RCFs). (A) GFP virus. RCFs were plated on a glass coverslip and infected with the GFP (top). Twenty-four hours after infection, the cells were fixed with 4% paraformaldehyde and viewed under fluorescent illumination. The fluorescent signal was detected in approximately 80% of cells infected with GFP. Cultures that were infected with a virus expressing an empty vector showed no detectable signal (data not shown). Magnification, ×200. (B) TαR virus. RCFs were plated and infected with either GFP or TαR virus, replated in 10-cm dishes, and grown to 80% confluence. The serum in the media was reduced to 0.1% for 18 to 24 hours, the cells were lysed, and 20 μg of cell lysate was subjected to Western blot analysis with an anti-αPDGFR or Ras-GAP (lysate control) antibody. Arrows: Full length (170-kDa) and truncated (100-kDa) receptors. Densitometric analysis revealed that the level of TαR was at least 50 times above the level of the endogenous full-length PDGFR.
Figure 1.
 
Retroviral expression of GFP and dominant negative PDGFα receptor (TαR) in primary rabbit conjunctival fibroblasts (RCFs). (A) GFP virus. RCFs were plated on a glass coverslip and infected with the GFP (top). Twenty-four hours after infection, the cells were fixed with 4% paraformaldehyde and viewed under fluorescent illumination. The fluorescent signal was detected in approximately 80% of cells infected with GFP. Cultures that were infected with a virus expressing an empty vector showed no detectable signal (data not shown). Magnification, ×200. (B) TαR virus. RCFs were plated and infected with either GFP or TαR virus, replated in 10-cm dishes, and grown to 80% confluence. The serum in the media was reduced to 0.1% for 18 to 24 hours, the cells were lysed, and 20 μg of cell lysate was subjected to Western blot analysis with an anti-αPDGFR or Ras-GAP (lysate control) antibody. Arrows: Full length (170-kDa) and truncated (100-kDa) receptors. Densitometric analysis revealed that the level of TαR was at least 50 times above the level of the endogenous full-length PDGFR.
Figure 2.
 
TαR attenuates PDGF-dependent contraction of RCFs. RCFs were infected with GFP or TαR and subjected to the collagen I gel contraction assay in the presence of buffer, 10% (vol/vol) FBS, or the indicated concentration of PDGF-AA. After 6 hours of exposure to the test substances, the extent of gel contraction was measured. Each experimental condition was assayed in triplicate and the mean ± SD is presented in the bar graph. *P < 0.05, **P < 0.01.
Figure 2.
 
TαR attenuates PDGF-dependent contraction of RCFs. RCFs were infected with GFP or TαR and subjected to the collagen I gel contraction assay in the presence of buffer, 10% (vol/vol) FBS, or the indicated concentration of PDGF-AA. After 6 hours of exposure to the test substances, the extent of gel contraction was measured. Each experimental condition was assayed in triplicate and the mean ± SD is presented in the bar graph. *P < 0.05, **P < 0.01.
Figure 3.
 
The retrovirus transduced GFP selectively into the cells of the epiretinal membrane. RCFs were prestained with red dye and injected into the rabbit’s eyes. The GFP virus was injected immediately afterward. Seven days later, the rabbit was killed, and the eye was removed, sectioned, and subjected to regular and fluorescence microscopic analysis. Epiretinal membrane under (A) regular illumination and (BD) UV illumination. (B) Injected cells (red fluorescein signal) and (C) infected cells (green signal). (D) Merge of (C) and (D). All micrographs are of approximately the same section. Magnification, ×200.
Figure 3.
 
The retrovirus transduced GFP selectively into the cells of the epiretinal membrane. RCFs were prestained with red dye and injected into the rabbit’s eyes. The GFP virus was injected immediately afterward. Seven days later, the rabbit was killed, and the eye was removed, sectioned, and subjected to regular and fluorescence microscopic analysis. Epiretinal membrane under (A) regular illumination and (BD) UV illumination. (B) Injected cells (red fluorescein signal) and (C) infected cells (green signal). (D) Merge of (C) and (D). All micrographs are of approximately the same section. Magnification, ×200.
Figure 4.
 
In vivo administration of the TαR attenuates PVR. Rabbits were subjected to gas vitrectomy and, after 3 days, cells, PRP, and virus were injected. Animals that were injected with GFP virus are designated as control, whereas the TαR group received the dominant negative PDGFR virus. The PVR was evaluated and scored on days 1, 4, 7, 14, 21, and 28 after surgery. The data for days 7 (left) and 28 (right) are presented. (•) Score for individual rabbits. The value at the bottom of each column is the percentage of animals that had retinal detachments (stage 3 or greater).
Figure 4.
 
In vivo administration of the TαR attenuates PVR. Rabbits were subjected to gas vitrectomy and, after 3 days, cells, PRP, and virus were injected. Animals that were injected with GFP virus are designated as control, whereas the TαR group received the dominant negative PDGFR virus. The PVR was evaluated and scored on days 1, 4, 7, 14, 21, and 28 after surgery. The data for days 7 (left) and 28 (right) are presented. (•) Score for individual rabbits. The value at the bottom of each column is the percentage of animals that had retinal detachments (stage 3 or greater).
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